Thawing methods and apparatus

Abstract

The invention provides a method of thawing a sample comprised in a container, the method comprising the steps of: a) calculating an agitation program as a function of either or both of the sample volume and the type of the container, and the ice fraction in the sample, and optionally the thermal conductivity of the sample container; b) agitating said sample according to the program to agitate at least one region of the sample; and c) changing the agitation program applied to at least one region of the sample in response to changes in the sample volume and / or sample ice fraction. The invention further provides a method of reducing shearing to cells during a method of agitation, and methods for thawing a sample wherein a sample container is differentially heated. An apparatus for use in the methods is also provided, as is an apparatus for thawing and / or cooling a sample which comprises a resilient vessel wall.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a National Stage application of PCT / GB2015 / 053720, filed Dec. 4, 2015 which claims the benefit of GB Application 1421556.0, filed on Dec. 4, 2014, both of which are incorporated by reference in their entirety herein.TECHNICAL FIELD OF INVENTION[0002]This invention relates to methods of thawing samples, in particular samples comprising biological material. The invention is further related to apparatus for thawing samples, especially samples comprising biological material.BACKGROUND TO THE INVENTION[0003]Cryopreservation is a widely employed technology used to maintain long term viability of biological samples for subsequent application in medicine, biotechnology and veterinary sciences. In order to obtain high viability upon thawing, it is necessary to add protective compounds (cryoprotectants) prior to freezing samples at a controlled cooling rate. Cryoprotectants dissolve in water in the sample and serve to prevent ic...

AI Technical Summary

Benefits of technology

[0039]Thus, the inventors have discovered that the amount of agitation of a sample to be thawed should be varied in accordance with its volume and its frozen or ice fraction, in order to minimise shearing of biological material within that sample and / or to decrease thaw times. In this respect, in a sample where 100% of the water is ice (or alternatively viewed, said sample has no liquid water), agitation may not be critical and thus may or may not be carried out. This is particularly the case when the sample is solid, i.e. comprises no free liquid. (For samples comprising only aqueous components, such samples may be solid when 100% of water is ice. For samples comprising, for example, cryoprotectants together with biological matter, these may be completely solid when vitrified, i.e. when at temperatures below the glass transition temperature of the cryoprotectant. Thus, the amount, type or frequency of agitation carried out at this point is not critical to the degree of shearing which may occur or to the thaw time of the sample. However, as the sample begins to thaw and a small amount of liquid water is present in the sample (e.g. at least 1, 2, 3, 4, 5, 10, 15 or 20% of the water is non-frozen or is liquid water, for example between 1-20% of the water is liquid water), vigorous agitation may be desirable. As the liquid water or non-frozen fraction of the sample increases (e.g. at least 50, 60, 70, 80 or 90% of the water in the sample is liquid water), it may be desirable to reduce the amount, or frequency of agitation, and thus to agitate the sample less vigorously. Typically, a sample that is vigorously agitated is agitated, e.g. by orbital rotation, at a frequency that is at least 1.5, 2, 3, 4, 5, 10, 50 or 100 times higher than that which is applied to a sample that has at least 50, 60, 70, 80, 90 or 100% liquid water. Less vigorous agitation refers to agitation which is at least 1.5, 2, 3, 4, 5, 10, or 100 fold lower than that which is applied to a sample with a small amount of liquid water present e.g. between 1-20% liquid water.

[0137]According to this aspect, the inventors have identified that the most detrimental period during the thawing process in terms of causing cell damage, which may affect cell viability, occurs during the thawing of the final portion of the ice fraction in a sample, e.g. the final 70% of the ice fraction. Generally, this final thaw occurs when the sample is heated at or above its Tm (e.g. at or above the Tm of the aqueous components e.g. water). During the initial heating phase, (e.g. the thawing of the initial 30% of the ice) cell damage is less likely to occur and thus this initial thaw can be carried out at a relatively slow rate, without having an effect on cell viability. The initial thaw is generally carried out a temperature which is equal to or greater than its Tg but is less than its Tm

Problems solved by technology

As discussed above, it is generally believed that, a slow rate of thawing is detrimental to the recovery of cells.

However, in practice this is not physically possible.

The primary underlying physical constraints in achieving rapid warming in the biological sample are associated with the thermal masses of the system, the enthalpy of fusion of the frozen material, and the thermal conductivities of the wall of the container and the liquid and solid phases of the sample.

The physics of thawing such a system leads to a relatively lengthy thawing period.

This is far from optimal in terms of cellular viability using current thawing methods.

In addition although the above described methods can reduce the thaw time of a sample it will be appreciated that when a sample material is heated via power / heat applied across a container wall, the material proximal to the wall will warm more quickly than that in the interior of the sample (i.e. with the greatest distance from a wall), which reduces heating power of the system.

However, although agitation can be used to decrease thaw times of samples, over agitating a sample can cause cellular damage by shearing.

In this regard, it is known that there is both a lower and upper limit for the amplitude and frequency that can be selected for a given geometry, where too high a frequency and or amplitude may result in overly agitating a sample such that the contents of the container are treated too harshly resulting in cellular damage (e.g. by excessive shearing).

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